Exploiting loop-grafting strategy resorting on computer-aided design to improve the thermostability of alpha-amylase from Geobacillus stearothermophilus

Abstract

Starch-based industries require a robust alpha-amylase capable of withstanding elevated temperatures and long incubation periods. In this study, a loop-grafting strategy based on a computer-aided design was proposed to engineer a thermostable alpha-amylase from Geobacillus stearothermophilus. Molecular dynamics (MD) simulations and trajectory analyses were employed to target the weak spots responsible for thermal inactivation, and three loop regions were identified as weak spots requiring improvement. Subsequently, substitute candidates from the thermophilic orthologous library were selected to refine these weak spots and the most promising mutant was screened using thermal unfolding and MD simulations. Expectedly, an alpha-amylase variant was constructed efficiently and specifically, which can withstand high-temperatures up to 100 °C without any stability-activity trade-off, giving an 8.0-fold longer half-life at 100 °C. Notably, the variant exhibited excellent thermotolerance during corn starch liquefaction at 100 °C, giving a 3.3-fold increased product concentration. Furthermore, the origin of the enhanced thermostability based on the dynamic trajectory of the variant was revealed. Successful attempts to tailor the thermal resistance and catalytic activity of alpha-amylase can further fuel the food industry, particularly high-temperature starch-based industries.